The present invention is generally in the area of media containing arabinogalactan for use in the cryopreservation of somatic cells.
Long-term storage of somatic cells and tissues is of widespread critical importance to the research and biomedical fields. Cryopreservation of cells and tissues is useful, for example, for the storage of blood products for clinical use; the establishment of organ banks; the long-term storage of cell lines to provide an unchanging population of cells; and the storage of populations of cells for research or medical purposes.
Somatic cells can be stored indefinitely once they reach liquid nitrogen temperature (-196.degree. C.). It has been well-established, however, that the freezing process itself results in immediate and long-term damage to cells with the greatest damage occurring to cells as they traverse the intermediate zone of temperature (-15.degree. C. to -60.degree. C.) during cooling and thawing (Mazur, Am. J. Physiol., 247:C125-142, 1984). The primary damaging physical events which can occur to cells during the process of freezing include dehydration and intracellular ice crystal formation. During freezing, solute is rejected from the solid phase producing an abrupt change in concentration in the unfrozen portion of solution. A biological cell responds to this perturbation by dehydrating to reach a new equilibrium state between intracellular and extracellular solutions. At high cooling rates, equilibrium cannot be maintained because the rate at which the chemical potential in the extracellular solution is being lowered is much greater than the rate at which water can diffuse out of the cell. The end result of this imbalance is that intracellular ice formation (IIF) is observed which is lethal to the cell. Toner, J. of Applied Phys., 67:1582-1593 (1990). At low cooling rates, cells are exposed for long periods of time at high subzero temperatures to high extracellular concentrations resulting in potentially damaging high intracellular concentrations. Lovelock, Biochem. Biophys. Acta, 10: 414-446 (1953).
The clinical and commercial application of cryopreservation for certain cell types is limited by the ability to recover a significant number of total viable cells. Significant losses in cell viability are observed in certain primary cell types. Examples of freeze-thaw cellular trauma have been encountered with cryopreservation of hepatocytes (Borel-Rinkes et al., Cell Transplantation, 1:281-292, 1992) porcine corneas (Hagenah and Bohnke, 30:396-406, 1993), bone marrow (Charak et al., Bone Marrow Transplantation, 11:147-154,1993) and porcine aortic valves (Feng et al., Eur. J. Cardiothorac. Surg., 6:251-255, 1992).
Cryopreservation protocols typically require the use of cryoprotective agents ("CPAs") to achieve clinically relevant survival rates for mammalian cells. A variety of substances have been used or investigated as potential additives to enhance survival of cells in the freezing process. The two most commonly used substances are glycerol and dimethyl sulfoxide. Other substances used include sugars, polymers, alcohols and proteins. CPAs can be divided roughly into two different categories; substances which permeate the cell membrane; and impermeable substances. One mechanism of protection results from reduction in the net concentration of ionic solutes for a subzero temperature when a CPA is present. This colligative effect is true for all substances which act as a CPA (Fahy, Biophys. J. 32:837-850, 1980). The addition of a CPA, however, changes the ionicity of the solution. Both tissues and intact organs can exhibit reduced cellular viability when exposed to sufficiently large step changes in external osmolarity produced by introduction of a freezing solution. Pegg, Cryobiology, 9:411-419 (1972). In addition, long term exposure to even low concentrations of certain CPAs at room temperature is potentially damaging (Fahy, Cryobiology, 27:247-268, 1990). Two of the most widely used cryopreservative agents, dimethylsulfoxide ("DMSO") and glycerol, are damaging to thawed cells due to osmotic complications and must be removed from cells post-thaw via rinsing and centrifugation.
Another media component routinely added to freezing media to reduce cell damage and death during freezing and thawing is serum. This additive, however, is highly complex and may add a number of factors (known and unknown) which may interfere with or alter cell function. Other non-permeating protective agents such as ethylene glycol, polyvinyl pyrrolidone (Klebe and Mancuso, In Vitro, 19:167-170, 1983) sucrose, and culture medium (Shier and Olsen, In Vitro Cell Dev. Biol., 31:336-337, 1995), have been studied for their effectiveness as cryoprotective agents for cells with variable results.
U.S. Pat. No. 4,004,975 to Lionetti et al. discloses the cryopreservation of leukocytes from centrifuged blood in a solution of hydroxyethyl starch and dimethylsulfoxide. U.S. Pat. No. 5,071,741 to Brockbank and PCT WO 92/08347 to Cryolife, published May 29, 1992, disclose the use of algae-derived polysaccharides such as agarose and alginate in a cryoprotective cell medium. U.S. Pat. No. 5,405,742 to Taylor discloses a solution for use as a blood substitute and for preserving tissue which includes dextran.
PCT WO 95/06068 (Abstract) discloses the use of polysaccharides in to improve hematopoietic functions and serve as a radioprotective agent. The use of gum arabic, cherry resin and apricot resin in ewe semen freezing medium is disclosed in Platov et al., Ovtsevodstvo, 10:38-39 (1980) (Abstract). Holtz et al., Proc. Fourth Intern. Symp. Repr. Phys. Fish, (1991) discloses the use of saccharides such as glucose and sucrose in the cryopreservation of trout semen. Hill et al., J. Lab. Clin. Med., 111:73-83 (1988) discloses the use of arabinogalactan to obtain washed murine platelets by centrifugation. Maisse, Aquat. Living Resour., 7:217-219 (1994) discloses a study of the effect of carbohydrates such as glucose and maltose on the cryopreservation of trout sperm. Isotonic sucrose in combination with calf serum has been used in a medium for the cryopreservation of mammalian cells. Shier and Olsen, In Vitro Cell. Dev. Biol., 31:336-337 (1995).
The preparation of derivatives of arabinogalactan and arabinogalactan degradation products is described in Prescott et al., Carbohydrate Research, 278:113-128 (1995); and U.S. Pat. No. 5,478,576 to Jung et al., the disclosures of which are incorporated herein by reference.
There is a need for additives which can be added to cell freezing media which stabilize cells during freezing, and protect cells from damage, which are non-toxic, and are suitable for a wide range of cell types in a wide variety of cell culture and clinical applications.
It is therefore an object of the invention to provide improved media for the preservation of somatic cells during freezing. It is a further object of the invention to provide media which can be used to preserve somatic cells and maintain the viability of the cells upon freezing and thawing. It is another object of the invention to provide media which can be used for the cryopreservation of a wide range of different somatic cell types.